The Class I histocompatibility ternary complex consists of three parts associated by noncovalent bonds. A transmembrane protein, called the MHC heavy chain is mostly exposed at the cell surface. The cell surface domains of the MHC heavy chain contain two segments of alpha helix that form two ridges with a groove between them. A short peptide binds noncovalently (“fits”) into this groove between the two alpha helices, and a molecule of beta-2 microglobulin binds noncovalently along side the outer two domains of the MHC monomer, forming a ternary complex. Peptides that bind noncovalently to one MHC subtype heavy chain usually will not bind to another subtype. However, all bind with the same type of beta-2 microglobulin. MHC molecules are synthesized and displayed by most of the cells of the body.
In humans, MHC molecules are referred to as HLA molecules. Humans primarily synthesize three different sub-types of MHC class 1 molecules designated HLA-A, HLA-B and HLA-C, differing only in the heavy chains.
The MHC works coordinately with a specialized type of T cell (the cytotoxic T cell) to rid the body of “nonself” or foreign viral proteins. The antigen receptor on T-cells recognizes an epitope that is a mosaic of the bound peptide and portions of the alpha helices of the making up the groove flanking it. Following generation of peptide fragments by cleavage of a foreign protein, the presentation of peptide fragments by the MHC molecule allows for MHC-restricted cytotoxic T cells to survey cells for the expression of “nonself” or foreign viral proteins. A functional T-cell will exhibit a cytotoxic immune response upon recognition of an MHC molecule containing bound antigenic peptide for which the T-cell is specific.
In the performance of these functions in humans, HLA-A, B, and C heavy chains interact with a multitude of peptides of about 8 to about 10, possibly about 8 to about 11, or about 8 to about 12 amino acids in length. Only certain peptides bind into the binding pocket in the heavy chain of each HLA sub-type as the monomer folds, although certain subtypes cross-react. By 1995, complete coding region sequences had been determined for each of 43 HLA-A, 89 HLA-B and 11 HLA-C alleles (P. Parham et al., Immunology Review 143:141-180, 1995).
Class II histocompatibility molecules consist of two transmembrane polypeptides that interact to form a groove at their outer end which, like the groove in class I molecules, non-covalently associates with an antigenic peptide. However, the antigenic peptides bound to class II molecules are derived from antigens that the cell has taken in from its surroundings. In addition, peptides that hind to class II histocompatibility molecules are 15 to about 25 or to about 30 amino acids in length. Only cells, such as macrophages, dendritic cells and B lymphocytes, that specialize in taking up antigen from extracellular fluids, express class II molecules.
It has long been thought that discovery of which antigen fragments will be recognized by class I MHC-restricted T-cells can lead to development of effective vaccines against cancer and viral infections. A number of approaches have been developed wherein algorithms are used to predict the amino acid sequence of HLA A, B, or C-binding peptides and several are available on the internet. For example, U.S. Pat. No. 6,037,135 describes a matrix-based algorithm that ranks peptides for likelihood of binding to any given HLA-A allele. Similarly, most prediction methods are limited to a set of alleles. Consequently, the predicted peptides may not bind to MHC monomers from a whole population of patients and thus may not be globally effective.
Another approach to identifying MHC-binding peptides uses a competition-based binding assay. All competition assays yield a comparison of binding affinities of different peptides. However, such assays do not yield an absolute dissociation constant since the result is dependent on the reference peptide used.
Still another approach used for determining MHC-binding peptides is the classical reconstitution assay, e.g. using “T2” cells, in which cells expressing an appropriate MHC allele are “stripped” of a native binding peptide by incubating at pH 2-3 for a short period of time. Then, to determine the binding affinity of a putative MHC-binding peptide for the same MHC allele, the stripped MHC monomer is combined in solution with the putative MHC-binding peptide, beta2-microglobulin and a conformation-dependent monoclonal antibody. The difference in fluorescence intensity determined between cells incubated with and without the test binding peptide after labeling, for example, either directly with the labeled monoclonal antibody or a fluorescence-labeled secondary antibody, can be used to determine binding of the test peptide. However, soluble MHC monomers stripped at low pH aggregate immediately, making their use in high through-put assays difficult and impractical.
There are currently a series of in vitro assays for cell mediated immunity which use cells from the donor. The assays include situations where the cells are from the donor, however, many assays provide a source of antigen presenting cells from other sources, e.g., B cell lines. These in vitro assays include the cytotoxic T lymphocyte assay; lymphoproliferative assays, e.g., tritiated thymidine incorporation; the protein kinase assays, the ion transport assay and the lymphocyte migration inhibition function assay (Hickling, J. K. et al, J. Virol., 61: 3463 (1987); Hengel, H. et al, J. Immunol., 139: 4196 (1987); Thorley-Lawson, D. A. et al, Proc. Natl. Acad. Sci. USA, 84: 5384 (1987); Kadival, G. J. et al, J. Immunol., 139: 2447 (1987); Samuelson, L. E. et el, J. Immunol., 139: 2708 (1987); Cason, J. et al, J. Immunol. Meth., 102: 109 (1987); and Tsein, R. J. et al, Nature, 293: 68 (1982)). These assays are disadvantageous in that they may lack true specificity for cell mediated immunity activity, they require antigen processing and presentation by an APC of the same MHC type, they are slow (sometimes lasting several days), and some are subjective and/or require the use of radioisotopes.
Yet another approach to identifying MHC class I-binding peptides utilizes formation of MHC tetramers, which are complexes of four MHC monomers with streptavidin, a molecule having tetrameric binding sites for biotin, to which is bound a fluorochrome, e.g., phycoerythrin. For class I monomers, soluble subunits of β2-microglobulin, the peptide fragment containing a putative T-cell epitope, and of a MHC heavy chain corresponding to the predicted MHC subtype of the peptide fragment of interest, are obtained by expression of the polypeptides in host cells. Each of the four monomers contained in the MHC tetramer is produced as a monomer by refolding these soluble subunits under conditions that favor assembly of the soluble units into reconstituted monomers, each containing a beta2-microglobulin, a peptide fragment, and the corresponding MHC heavy chain. An MHC tetramer is constructed from the monomers by biotinylation of the monomers and subsequent contact of the biotinylated reconstituted monomers with fluorochrome-labeled streptavidin. When contacted with a diverse population of T cells, such as is contained in a sample of the peripheral blood lymphocytes (PBLs) of a subject, those tetramers containing reconstituted monomers that are recognized by a T cell in the sample will bind to the matched T cell. Contents of the reaction is analyzed using fluorescence flow cytometry, to determine, quantify and/or isolate those T-cells having a MHC tetramer bound thereto (See U.S. Pat. No. 5,635,363).
At least one other test is required to determine whether a test peptide recognized by a T-cell by the MHC tetramer assay will activate the T-cell to generate an immune response, a so-called “functional test”. The enzyme-linked immunospot (ELISpot) assay has been adapted for the detection of individual cells secreting specific cytokines or other effector molecules by attachment of a monoclonal antibody specific for a cytokine or effector molecule on a microplate. Cells stimulated by an antigen are contacted with the immobilized antibody. After washing away cells and any unbound substances, a tagged polyclonalantibody or more often, a monoclonal antibody, specific for the same cytokine or other effector molecule is added to the wells. Following a wash, a colorant that binds to the tagged antibody is added such that a blue-black colored precipitate (or spot) forms at the sites of cytokine localization. The spots can be counted manually or with automated ELISpot reader system to quantitated the response. A final confirmation of T-cell activation by the test peptide may require in vivo testing, for example in a mouse model. Thus, the route to final confirmation of the efficacy of a MHC-binding peptide is expensive and time consuming.
Thus, there is still a need in the art for new and better systems and methods for preliminary screening assays identifying putative MHC class I-binding peptides and for measuring peptide binding to MHC class I alleles, such as HLA-A, B or C, especially an in vitro assay in solid phase format. There is also a need in the art to develop methods to determine the MHC-binding affinity of MHC-binding peptides and for a measurement for the dissociation rate of a bound peptide from the MHC molecule.